Xu Fei-Fei, Liu Xiu-Hua, Zhu Xiao-Mei
Department of Pathophysiology, Chinese PLA General, Hospital, Beijing 100853, China.
Sheng Li Xue Bao. 2008 Feb 25;60(1):29-37.
The present study was aimed to investigate whether calreticulin (CRT) was involved in the protective effect of hypoxic preconditioning (HPC) against oxidative stress injury in rat cardiomyocytes. Neonatal cardiomyocytes were prepared from Sprague-Dawley rats aged 24 h, and cultured in DMEM medium containing 10% fetal bovine serum. The cultured cardiomyocytes were randomly divided into 8 groups as follows: (1) hydrogen peroxide stress (H(2)O(2) group); (2) brief hypoxic exposure for 20 min to simulate HPC (HPC group); (3) hypoxic exposure for 20 min followed by normoxic reoxygenation for 24 h before hydrogen peroxide stress (HPC + H(2)O(2) group); (4) SB203580 (a specific inhibitor of p38 MAPK) + HPC + H(2)O(2) group; (5) CRT antisense oligonucleotide transfection (AS group); (6) AS + H(2)O(2) group; (7) AS + HPC + H(2)O(2) group; (8) control group. Morphological observation, lactate dehydrogenase (LDH) leakage and flow cytometry were employed to assess cell apoptosis and necrosis. RT-PCR and Western blot were used to detect CRT expression and activity of p38 MAPK. All experiments were repeated at least four separate times. The results obtained were as follows: (1) HPC relieved cell injury caused by H(2)O(2). Compared with that in H(2)O(2) group, the cell survival rate increased by 18.0% (P<0.05), apoptotic rate and LDH leakage in culture medium decreased by 19.4% and 53.0%, respectively (P<0.05) in HPC + H(2)O(2) group. (2) H(2)O(2) induced CRT over-expression (7.1-fold increase compared with control, P<0.05), while HPC resulted in mild CRT up-regulation (2.4-fold increase compared with control, P<0.05), suggesting that HPC can relieve the over-expression of CRT induced by H(2)O(2). (3) CRT AS transfection weakened the protection of HPC. Compared with that in HPC + H(2)O(2) group, the cell survival rate decreased by 4% (P<0.05), and apoptotic rate and LDH leakage in culture medium increased by 2.6% and 39.0%, respectively (P< 0.05) in AS + HPC + H(2)O(2) group. (4) The protection of HPC and HPC-induced upregulation of CRT were almost eliminated when SB203580 was administered before HPC. These results suggest that HPC up-regulates CRT expression through the p38 MAPK signaling pathway and protects cardiomyocytes from oxidative stress injury.
本研究旨在探讨钙网蛋白（CRT）是否参与低氧预处理（HPC）对大鼠心肌细胞氧化应激损伤的保护作用。从24小时龄的Sprague-Dawley大鼠制备新生心肌细胞，并在含有10%胎牛血清的DMEM培养基中培养。将培养的心肌细胞随机分为8组如下：（1）过氧化氢应激组（H₂O₂组）；（2）短暂低氧暴露20分钟以模拟HPC组（HPC组）；（3）低氧暴露20分钟，然后在过氧化氢应激前常氧复氧24小时组（HPC + H₂O₂组）；（4）SB203580（p38丝裂原活化蛋白激酶的特异性抑制剂）+ HPC + H₂O₂组；（5）CRT反义寡核苷酸转染组（AS组）；（6）AS + H₂O₂组；（7）AS + HPC + H₂O₂组；（8）对照组。采用形态学观察、乳酸脱氢酶（LDH）漏出及流式细胞术评估细胞凋亡和坏死。采用逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法检测CRT表达及p38丝裂原活化蛋白激酶活性。所有实验至少独立重复4次。获得的结果如下：（1）HPC减轻了H₂O₂所致的细胞损伤。与H₂O₂组相比，HPC + H₂O₂组细胞存活率提高了18.0%（P<0.05），凋亡率及培养基中LDH漏出分别降低了19.4%和53.0%（P<0.05）。（2）H₂O₂诱导CRT过表达（与对照组相比增加7.1倍，P<0.05），而HPC导致CRT轻度上调（与对照组相比增加2.4倍，P<0.05），提示HPC可减轻H₂O₂诱导的CRT过表达。（3）CRT反义寡核苷酸转染减弱了HPC的保护作用。与HPC + H₂O₂组相比，AS + HPC + H₂O₂组细胞存活率降低了4%（P<0.05），凋亡率及培养基中LDH漏出分别增加了2.6%和39.0%（P<0.05）。（4）在HPC前给予SB203580时，HPC的保护作用及HPC诱导的CRT上调几乎被消除。这些结果提示，HPC通过p38丝裂原活化蛋白激酶信号通路上调CRT表达并保护心肌细胞免受氧化应激损伤。
